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\begin{document}

\title{Auxotrophs compromise bio-production in yeast Saccharomyces cerevisiae}



\author[1]{Chunxiao Yan}%
\author[1]{Ning Gao}%
\author[1]{Xuan Cao}%
\author[1]{Lun Yao}%
\author[2]{Yongjin Zhou}%
\author[3]{Jiaoqi Gao}%
\affil[1]{Chinese Academy of Sciences Dalian Institute of Chemical Physics}%
\affil[2]{Dalian Institute of Chemical Physics}%
\affil[3]{Dalian Institute of Chemical Physics Department of Biotechnology}%


\vspace{-1em}



  
  \date{May 04, 2024}


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\selectlanguage{english}
\begin{abstract}
Auxotrophic marker genes have widely used for genetic engineering in
yeast. However, the effects of auxotrophic strains that are deficient in
synthesis of amino acids or nucleotides on the growth and production are
rarely reported. In this study, a total of eight auxotrophic strains
with single knockout of selective markers were obtained to evaluate cell
growth and free fatty acid (FFA) production in Saccharomyces cerevisiae
with supplementing different concentrations of amino acids or
nucleotides. Generally, except for gene ADE2, most auxotrophic strains
possessed decreased cell growth and FFA production, which could be
remedied by the higher concentrations of supplements. leu2\selectlanguage{greek}Δ \selectlanguage{english}damaged both
growth and production even with supplementation of 1000 mg/L leucine.
Therefore, this study shows that auxotrophs compromise the metabolic
engineering endeavor and provides a guidance in supplementing amino
acids or nucleotides during fermentations for maximizing
bio-productions.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

Bio-manufacturing via microbial cell factories represents for an
alternative production route for compounds previously obtained from
natural resources or chemical synthesis \textsuperscript{{[}1,2{]}},
considering advantages in climate insensitivity, low land occupancy and
environmental friendliness \textsuperscript{{[}3{]}}. At present, the
production of bulk chemicals, organic acids \textsuperscript{{[}4{]}},
fatty alcohols\textsuperscript{{[}5{]}}, and alkanes
\textsuperscript{{[}6{]}} have been realized in microbes. In addition,
with the help of developed technologies of systems biology, protein
engineering, cofactor engineering and computer-aided modeling
\textsuperscript{{[}7,8{]}}, complex biosynthetic pathways have been
designed to enable the productions of compounds like cannabidiol
\textsuperscript{{[}9{]}} and scopolamine \textsuperscript{{[}10{]}}. In
particular, the industrial-scale productions of artemisinin
\textsuperscript{{[}11{]}}, farnesene \textsuperscript{{[}12{]}} and
squalene\textsuperscript{{[}13{]}} demonstrate the greatest potential of
microbial cell factories.

The auxotrophic marker gene is wildly used for convenient genetic
manipulation during cell factory construction. However, the auxotrophic
genotype might compromise the bio-production due to the insufficient
supply of nutrients. For example, the auxotroph is often constructed by
disrupting some essential genes for amino acids synthesis, such as
leucine (\emph{LEU2} ), histidine (\emph{HIS3} ) and uracil (\emph{URA3}
) for convenient genetic manipulation in yeast\emph{Saccharomyces
cerevisiae} \textsuperscript{{[}14{]}}. Traditional genetic manipulation
relies on the insertion of heterologous metabolic pathways into the
locus of auxotrophic markers \textsuperscript{{[}15{]}}. Despite the
fast development of molecular biology, auxotrophic selective markers are
still used to maintain plasmid stability, and even sgRNA expression in
the CRISPR/Cas9 system \textsuperscript{{[}16{]}}. However, amino acids,
as the precursors of protein synthesis, participate in various metabolic
pathways and are crucial for microbial growth and
production\textsuperscript{{[}17{]}}. Therefore, the exogenous addition
of amino acids during fermentations \textsuperscript{{[}15{]}}, may be
not enough to support efficient cell growth to decrease product yields,
and even misjudge the feasibility of metabolic engineering
strategy\textsuperscript{{[}18{]}}, which, however, has not been paid
much attentions by researchers.

Here, we evaluated the effects of eight commonly used auxotrophic
markers, with grouping them into synthesis of acetyl-CoA,
\selectlanguage{greek}α-\selectlanguage{english}ketoglutarate, and succinyl-CoA derived modules (Figure 1), in cell
growth and bio-productions of free fatty acid (FFA) in \emph{S.
cerevisiae} \textsuperscript{{[}19{]}}. By comparing the prototrophic
and auxotrophic strains with exogenous supplements of different
concentrations of amino acids or nucleotides, we concluded that except
for gene \emph{ADE2} , most auxotrophic strains possessed decreased cell
growth and FFA production, which could be remedied by the higher
concentrations of supplements. \emph{leu2} \selectlanguage{greek}Δ \selectlanguage{english}damaged both growth and
production even in 1000 mg/L of supplemented leucine, which is not
suitable as a screening marker. This study reminded us to be cautious to
use auxotrophic cell factory in bio-productions, and the recovery of
these genes in fermentations is supposed to be essential for enhancing
product yields.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Strains and media}

All strains and plasmids used in this study were listed in supplementary
tables S1 and S2, respectively. \emph{S. cerevisiae} were cultivated in
YPD medium containing 10 g/L yeast extract, 20 g/L peptone and 20 g/L
glucose. \selectlanguage{greek}\emph{Escherichia coli DH5α} \selectlanguage{english}was grown in LB medium (5 g/L
yeast extract, 10 g/L NaCl and 10 g/L tryptone), and 100 mg/mL
ampicillin was added. Synthetic Dropout (SD) medium was composed of 20
g/L glucose and 6.7 g/L amino acid-free yeast nitrogen source (YNB).
Minimal (Delft) medium was adopted to cultivate \emph{S.
cerevisiae}strains \textsuperscript{{[}20{]}} containing 20 g/L glucose,
2.5 g/L (NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4}, 14.4
g/L KH\textsubscript{2}PO\textsubscript{4}, 0.5 g/L
MgSO\textsubscript{4}*7H\textsubscript{2}O, 2 ml/L trace metals, 1 ml/L
vitamin solution, and amino acids were supplemented if necessary. Solid
plates contain 20 g/L agar.

The specific amounts of exogenous amino acids or nucleotides added in
media are as follows. In preliminary experiments, low (20-60 mg/L,
Figure S2) and high (100 mg/L, Figure S3) concentrations of amino acids
or nucleotides were added in the culture medium, respectively, to
determine the growth and production of the auxotrophic strains.
Furthermore, we set 100 mg/L as the baseline to test the essential
amounts of amino acids and nucleotides for each auxotroph to restore
cell growth and bio-productions. For example, the concentrations of
leucine, tryptophan, lysine (Figure 2) and methionine (Figure 3) were
increased from 100 mg/L to 500 and 1,000 mg/L, respectively. The
concentrations of histidine, arginine (Figure 4), uracil and adenine
(Figure 5) were decreased from 100 mg/L to 20 and 60 mg/L.

\textbf{2.2 Construction of engineered strain}

All \emph{S. cerevisiae} strains used in this study were derived from
CEN.PK113-11C (\selectlanguage{greek}\emph{MATa SUC2 MAL2-8c his3Δ1 ura3-52}\selectlanguage{english} ). The primers
used in this study were listed in Table S3. Firstly, strain YHM01 was
obtained via integrating \emph{Cas9} gene expression box at neutral site
XI of FFA producing strain YJZ06 \textsuperscript{{[}19{]}}. ScYan01
(\emph{ura3} \selectlanguage{greek}Δ\selectlanguage{english}) and ScYan08 (\emph{his3} \selectlanguage{greek}Δ\selectlanguage{english}) were obtained by \emph{in
situ} restoration of gene \emph{HIS3} and \emph{URA3} in strain YHM01,
respectively. Furthermore, other auxotrophic strains were constructed
based on ScYan01, adopting the CRISPR/Cas9 system with different sgRNA
and donor DNA (approximately 500 bp upstream and downstream homologous
arms) \textsuperscript{{[}16{]}}, obtaining engineered strains with
double deficient genes of \emph{URA3} and other screening marker.
Finally, the single auxotrophic strains were obtained by \emph{in
situ}repair of \emph{URA3} gene. Gene \emph{HIS3} was \emph{in
situ}supplemented in strain ScYan08, generating strain ScYan09 as the
positive control, without any deletions of auxotrophic genes. The
obtained engineering strains were tested on SD + / - amino acid or
nucleotide (20 mg/L) culturing at 30 for 2 - 3 days, as shown in Figure
S1.

\textbf{2.3 Growth curve measurement}

Strains were inoculated with initial OD\textsubscript{600} of 0.2 in
Delft minimal medium, supplementing different concentrations of amino
acids or nucleotide. Samples were taken at 0 h, 6 h, 12 h, 24 h, 36 h
and 48 h, respectively, and the optical density was measured at 600 nm
(OD\textsubscript{600}) by visible light spectrophotometer. Samples were
diluted to OD\textsubscript{600} = 0.2 - 0.8 for measurement, and
OD\textsubscript{600} values were calculated to demonstrate cell growth.

\textbf{2.4 Quantification of free fatty acids}

All strains were precultured in YPD medium for 24 h and then transferred
to Delft minimal medium containing 20 g/L glucose and specific amino
acids or nucleotides at 30, 220 rpm. After cultivation for 48 h, FFA
were determined according to the previous
method\textsuperscript{{[}19{]}}. In brief, 100 \selectlanguage{greek}μ\selectlanguage{english}L sample was taken for
FFA extraction. Then, the 100 \selectlanguage{greek}μ\selectlanguage{english}L ddH\textsubscript{2}O, 10 \selectlanguage{greek}μ\selectlanguage{english}L 40\%
tetrabutylammonium hydroxide and 200 \selectlanguage{greek}μ\selectlanguage{english}L dichloromethane (200 mM methyl
iodide as methyl donor and 100 mg/L pentadecanoic acid as an internal
standard) were successively added to the sample. The mixtures were
shaken for 30 minutes at 1,600 rpm in whirlpool mixer, and
centrifugation was performed at 3,000 g for 10 minutes to promote phase
separation. The 200 \selectlanguage{greek}μ\selectlanguage{english}L dichloromethane was transferred to GC bottles
containing glass insert. The samples were dried at room temperature for
4 h, and re-suspended with 200 \selectlanguage{greek}μ\selectlanguage{english}L N-hexane. Finally, the samples were
analyzed by gas chromatography. Specific procedures is shown by
predecessors \textsuperscript{{[}19{]}}.

\textbf{3 RESULTS}

\textbf{3.1 Auxotrophs of \emph{S. cerevisiae} decreased both cell
growth and FFA production}

Eight auxotrophs that are deficient in synthesis of some specific amino
acids and nucleotides, including \emph{ura3} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{his3}
\selectlanguage{greek}Δ,\selectlanguage{english}\emph{met15} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{leu2} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{trp1} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{ade2} \selectlanguage{greek}Δ,
\selectlanguage{english}\emph{arg4} \selectlanguage{greek}Δ, \selectlanguage{english}and \emph{lys2} \selectlanguage{greek}Δ, \selectlanguage{english}were constructed in an FFA producing
strain YJZ06\textsuperscript{{[}19{]}}. These strains grew normally in
minimal plates supplementing the corresponding nutrition (Figure S1).
While cultivating in minimal media containing commonly used
concentrations of 20 mg/L of uracil and histidine, 60 mg/L of
leucine\textsuperscript{{[}14{]}}, 20 mg/L of methionine and
arginine\textsuperscript{{[}21{]}}, 40 mg/L of
tryptophan\textsuperscript{{[}22{]}}, 30 mg/L of lysine
\textsuperscript{{[}23{]}}and 50 mg/L of adenine
\textsuperscript{{[}24{]}}, a serious decreased growth and FFA titers
were observed. In particular, \emph{ura3} \selectlanguage{greek}Δ,\selectlanguage{english}\emph{met15} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{leu2}
\selectlanguage{greek}Δ, \selectlanguage{english}\emph{arg4} \selectlanguage{greek}Δ, \selectlanguage{english}and \emph{lys2} \selectlanguage{greek}Δ \selectlanguage{english}strains had 13.5-35.0\% of maximum
biomass and 23.7-44.2\% of FFA in compared with the prototrophic strain
(Figure S2). In this case, we suspected that the supplemented amino
acids and nucleotides were not enough to support cell growth and FFA
production. We increased the concentrations of amino acids and
nucleotides to 100 mg/L to cultivate the strains (Figure S3). Growth
behaviors of strains like \emph{ura3} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{his3} \selectlanguage{greek}Δ,\selectlanguage{english}\emph{arg4} \selectlanguage{greek}Δ,
\selectlanguage{english}and \emph{ade2} \selectlanguage{greek}Δ \selectlanguage{english}had been greatly improved with a slight influence on
FFA titers, but strains of \emph{leu2} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{met15} \selectlanguage{greek}Δ,\selectlanguage{english}\emph{trp1} \selectlanguage{greek}Δ,
\selectlanguage{english}and \emph{lys2} \selectlanguage{greek}Δ \selectlanguage{english}still grew poorly with an extremely low FFA titers,
which demonstrated that effects of auxotrophic strains were
distinguished, and different concentrations of amino acids and
nucleotides were required. Therefore, we set 100 mg/L as the baseline to
test the essential amounts of amino acids and nucleotides to restore
cell growth and bio-productions.

\textbf{3.2 Large amounts of required amino acids that derived
acetyl-CoA and succinyl-CoA}

The six amino acids are divided into three groups according to their
specific intermediates in the tricarboxylic acid (TCA) cycle, as
acetyl-CoA module (leucine, tryptophan, lysine), succinyl-CoA module
(methionine), and \selectlanguage{greek}α-\selectlanguage{english}ketoglutarate module (histidine, arginine) (Figure
1). As shown in Figure S2, 100 mg/L of leucine, tryptophan, and lysine
could not support efficient cell growth, and hence the concentrations
were further increased to 500 mg/L and 1,000 mg/L. As expected, to
restore cell growth and FFA production, the required amounts of amino
acids in acetyl-CoA module are extremely high, up to 1,000 mg/L (Figure
2). For strains deficient in synthesis of tryptophan and lysine, the
cell growth and production capacity were basically similar to those of
control strain with 500 mg/L of supplements. However, compared with
control strain, only 70\% of maximum biomass and FFA titers was obtained
even in 1,000 mg/L of leucine (Figure 2A, D), demonstrating leucine
metabolism may be closely related to acetyl-CoA for cell growth and
product formation.

Similar to those in acetyl-CoA module, methionine in succinyl-CoA module
also needs higher concentration to restore cell growth. While
supplementing 500 mg/L and 1,000 mg/L of methionine to cultivate the
auxotrophic strain, the growth and FFA were similar to the control
strain, which was obviously improved than those in 100 mg/L (Figure 3).
The degradation of methionine generates succinyl-CoA through a
multi-step reaction involved in intermediates of methylmalonyl-coA and
methylmalonyl-SCOA (Figure 1), whose absence may disturb central
metabolism for cell growth and production.

\selectlanguage{greek}\textbf{3.3 Small amounts of required amino acids in α-ketoglutarate
module}\selectlanguage{english}

The metabolism of arginine and histidine is involved in the synthesis of
the intermediate \selectlanguage{greek}α-\selectlanguage{english}ketoglutarate of TCA cycle (Figure 1), which also
showed relatively small amounts of required supplements to restore cell
growth (Figure S3). Therefore, the lower concentrations of arginine and
histidine (20, 60, and 100 mg/L) were adopted to cultivated the
auxotrophic strains to evaluate their roles in cell growth and FFA
production (Figure 4).

\emph{his3} \selectlanguage{greek}Δ \selectlanguage{english}had little impact on cell growth and FFA production, and
the auxotrophic strain reached the optimal behavior of control strain in
60 mg/L of histidine. The performance of strain did not continue to
improve when the concentration was further increased to 100 mg/L.
Different from histidine, while supplementing 60 mg/L of arginine, the
OD\textsubscript{600} and FFA titer were only 60\% and 30\% of those in
control strain within 48 h, respectively (Figure 4B, D). The minimal
required amount of arginine was approximately 100 mg/L.

\textbf{3.4 Effects of nucleotides on growth and production}

Except for amino acids, the deficient synthesis of nucleotides (uracil
and adenine) is also commonly used screening markers. The absence or
insufficiency of nucleotides will inevitably affect cell growth,
considering their essential roles in translation process as three
nucleotide codons in the central rule. We investigated the effects of
exogenous addition of nucleotides with different concentrations on the
growth and production in two nucleotide deficient strains (\emph{ura3} \selectlanguage{greek}Δ
\selectlanguage{english}and \emph{ade2} \selectlanguage{greek}Δ\selectlanguage{english}). As 100 mg/L of supplements are sufficient (Figure
S3), lower concentrations of 20, 60, and 100 mg/L were utilized to
determine the growth and production capacity of the strains (Figure 5).

These two deficient strains possessed a similar characteristic in
required amounts of nucleotides. The performances of the strains were
similar to the control strain when only 60 mg/L uracil or adenine was
added, and the increased concentrations did not further improved cell
growth (Figure 5A, B). FFA titers of the strain \emph{ura3} \selectlanguage{greek}Δ
\selectlanguage{english}supplemented with 60 mg/L and 100 mg/L uracil were about 90\% of that in
control strain (Figure 5C). In contrast, FFA titers of strain\emph{ade2}
\selectlanguage{greek}Δ \selectlanguage{english}were 60\% and 90\% of that of control strain, respectively (Figure
5D).

\textbf{4. DISCUSSION}

Auxotrophic genes are commonly applied as screening markers in genetic
manipulation of \emph{S. cerevisiae} , but their negative roles in
bio-productions in metabolic engineering are often overlooked. The
deficient synthesis of amino acids or nucleotides may interfere with the
homeostasis of microbial cell and hinder cell
growth\textsuperscript{{[}18{]}}. In this study, eight commonly used
auxotrophic makers were selected to investigate their effects on cell
growth and FFA production in \emph{S. cerevisiae} .

The six amino acids are divided into three groups based on their
synthesis and degradation metabolism. Generally, except gene\emph{ADE2}
, all these markers decreased both cell growth and FFA production of the
corresponding auxotrophs under the commonly used concentrations.
Interestingly, these observed repressions could be remedied by the much
higher concentrations of supplements than expected ([?]500 mg/L), which
guided us to increase the supplemented concentrations while adopting
auxotrophs. Among them, the minimal required amounts of amino acids from
acetyl-CoA and succinyl-CoA modules are supposed to be much higher than
those from \selectlanguage{greek}α-\selectlanguage{english}ketoglutarate module and nucleotides (500 mg/L vs 60 mg/L),
which may be attributed to the competition with cell growth. As
reported, the strain \emph{ade2} \selectlanguage{greek}Δ \selectlanguage{english}will turn red under limited
concentrations, which is suitable for high-throughput
screening\textsuperscript{{[}25{]}}.

In particular, deficiency in leucine synthesis greatly decreased both
cell growth and FFA production, even in medium supplementing 1,000 mg/L
of leucine, as observed in other species \textsuperscript{{[}26-29{]}}.
There is a close relationship of gene \emph{LEU2} with cell growth and
lipogenesis, which has previously been reported that leucine metabolism,
but not leucine itself played a key role as a signal to affect TOR
activity. Here, leucine metabolism was inferred as an alternative way to
offer cytoplasmic acetyl-CoA to regulate the cell growth and FFA
production. Therefore, the auxotrophic strains may be not suitable for
bio-productions, especially in the long-run fermentations.

\textbf{5 CONCLUSION}

In summary, it is not desirable to disrupt the synthesis of amino acids
or nucleotides in order to construct a heterologous metabolic pathway in
metabolic engineering. Currently, the optimized CRISPR-Cas9 gene editing
tool may provide an alternative solution to achieve the recovery and
reuse of screening markers \textsuperscript{{[}16{]}}. Meanwhile,
numerous neutral loci in \emph{S. cerevisiae} have been characterized
for multiple gene integration \textsuperscript{{[}30{]}}. The recovery
of auxotrophic markers to generate the prototrophic strain is supposed
to be essential to maximize bio-productions during the fermentation, and
alleviate the interference of internal environment for the engineered
strains.

\textbf{Acknowledgments}

We thank Dr. Ruibing Chen (Naval Medical University) for kindly sharing
the plasmid (sgRNAs contain amino acids or nucleotides corresponding
gene recognition sites). This study was financially supported by
National Key Research and Development Program of China (2021YFC2104200),
and National Natural Science Foundation of China (21877111).

\textbf{Conflict of interest:} The authors declare no competing
interests

\textbf{Data availability}

All related data and information are available from the corresponding
author on reasonable request

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27 Rodriguez-Frometa, R. A., Gutierrez, A., Torres-Martinez, S. \&
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\textbf{Figure 1 Synthesis and degradation of amino acids are closely
related to cell growth and FFA production.} Six amino acids are grouped
as acetyl-CoA module (lysine, tryptophan, leucine), succinyl-CoA module
(methionine), and \selectlanguage{greek}α-\selectlanguage{english}ketoglutarate module (histidine, arginine). The
investigated amino acids are colored in red, and compounds marked in
orange are intermediates of amino acids involved in the tricarboxylic
acid (TCA) cycle.

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\verb`image2.emf`  available at \url{https://authorea.com/users/513950/articles/589943-auxotrophs-compromise-bio-production-in-yeast-saccharomyces-cerevisiae}

\textbf{Figure 2 Effects of auxotrophic genes from acetyl-CoA module on
cell growth and FFA production.} Control: prototrophic
strain;\emph{leu2} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan11 with single deletion of gene
\emph{LEU2} ;\emph{trp1} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan12 with single deletion of gene
\emph{TRP1} ;\emph{lys2} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan14 with single knocked out gene
\emph{LYS2} . Growth curve of \emph{leu2} \selectlanguage{greek}Δ, \selectlanguage{english}\emph{trp1} \selectlanguage{greek}Δ \selectlanguage{english}and
\emph{lys2} \selectlanguage{greek}Δ \selectlanguage{english}were measured by adding exogenous leucine, tryptophan and
lysine of 100, 500 and 1,000 mg/L, respectively (A, B and C). Meanwhile,
fatty acid titers at fermentation endpoint (48 h) of \emph{leu2} \selectlanguage{greek}Δ,
\selectlanguage{english}\emph{trp1} \selectlanguage{greek}Δ \selectlanguage{english}and\emph{lys2} \selectlanguage{greek}Δ \selectlanguage{english}were measured (D). All data was
represented as the mean \selectlanguage{ngerman}± s.d. of three clone samples.

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\verb`image3.emf`  available at \url{https://authorea.com/users/513950/articles/589943-auxotrophs-compromise-bio-production-in-yeast-saccharomyces-cerevisiae}

\textbf{Figure 3 Effects of auxotrophic genes from succinyl-CoA module
on cell growth and FFA production.} Control: prototrophic
strain;\emph{met15} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan10 with single deletion of gene
\emph{MET15} . Growth curve (A) and fatty acid titers (B) at
fermentation endpoint (48 h) of \emph{met15} \selectlanguage{greek}Δ \selectlanguage{english}were measured by adding
exogenous methionine of 100, 500 and 1,000 mg/L, respectively. All data
was represented as the mean \selectlanguage{ngerman}± s.d. of three clone samples.

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\verb`image4.emf`  available at \url{https://authorea.com/users/513950/articles/589943-auxotrophs-compromise-bio-production-in-yeast-saccharomyces-cerevisiae}

\selectlanguage{greek}\textbf{Figure 4 Effects of auxotrophic genes from α-Ketoglutarate
module on cell growth and FFA production.}\selectlanguage{english} Control: prototrophic
strain;\emph{his3} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan8 with single damage of gene
\emph{HIS3} ;\emph{arg4} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan13 with single removal of gene
\emph{ARG4} . Growth curve of \emph{his3} \selectlanguage{greek}Δ \selectlanguage{english}and \emph{arg4} \selectlanguage{greek}Δ \selectlanguage{english}were
measured by adding exogenous histidine and arginine of 20, 60 and 100
mg/L, respectively (A and B). Fatty acid titers at fermentation endpoint
(48 h) of\emph{his3} \selectlanguage{greek}Δ \selectlanguage{english}and \emph{arg4} \selectlanguage{greek}Δ \selectlanguage{english}were measured (C and D). All
data was represented as the mean \selectlanguage{ngerman}± s.d. of three clone samples.

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\verb`image5.emf`  available at \url{https://authorea.com/users/513950/articles/589943-auxotrophs-compromise-bio-production-in-yeast-saccharomyces-cerevisiae}

\textbf{Figure 5 Effects of auxotrophic genes from nucleotide module on
cell growth and FFA production.} Control: prototrophic
strain;\emph{ura3} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan1 with single damage of gene
\emph{URA3} ;\emph{ade2} \selectlanguage{greek}Δ: \selectlanguage{english}strain ScYan15 with single deletion of gene
\emph{ADE15} . Growth curve of strain \emph{ura3} \selectlanguage{greek}Δ \selectlanguage{english}and \emph{ade2} \selectlanguage{greek}Δ
\selectlanguage{english}were measured by adding exogenous histidine of 20, 60 and 100 mg/L,
respectively (A and B). Fatty acid titers at fermentation endpoint (48
h) of \emph{ura3} \selectlanguage{greek}Δ \selectlanguage{english}and \emph{ade2} \selectlanguage{greek}Δ \selectlanguage{english}were measured (C and D). All data
was represented as the mean \selectlanguage{ngerman}± s.d. of three clone samples.

\selectlanguage{english}
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