PMID- 29753070
OWN - NLM
STAT- MEDLINE
DCOM- 20190722
LR  - 20220331
IS  - 1096-7184 (Electronic)
IS  - 1096-7176 (Linking)
VI  - 48
DP  - 2018 Jul
TI  - Iterative algorithm-guided design of massive strain libraries, applied to 
      itaconic acid production in yeast.
PG  - 33-43
LID - S1096-7176(18)30118-6 [pii]
LID - 10.1016/j.ymben.2018.05.002 [doi]
AB  - Metabolic engineering requires multiple rounds of strain construction to evaluate 
      alternative pathways and enzyme concentrations. Optimizing multigene pathways 
      stepwise or by randomly selecting enzymes and expression levels is inefficient. 
      Here, we apply methods from design of experiments (DOE) to guide the construction 
      of strain libraries from which the maximum information can be extracted without 
      sampling every possible combination. We use Saccharomyces cerevisiae as a host 
      for a novel six-gene pathway to itaconic acid, selected by comparing alternative 
      shunt pathways that bypass the mitochondrial TCA cycle. The pathway is 
      distinctive for the use of acetylating acetaldehyde dehydrogenase to increase 
      cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the 
      cytosol, and an itaconic acid exporter. Precise control over the expression of 
      each gene is enabled by a set of promoter-terminator pairs that span a 174-fold 
      range. Two large combinatorial libraries (160 variants, 2.4 Mb and 32 variants, 
      0.6 Mb) are designed where the expression levels are selected by statistical 
      methods (I-optimal response surface methodology, full factorial, or 
      Plackett-Burman) with the intent of extracting different types of guiding 
      information after the screen. This is applied to the design of a third library 
      (24 variants, 0.5 Mb) intended to alleviate a bottleneck in cis-aconitate 
      decarboxylase (CAD) expression. The top strain produces 815 mg/l itaconic acid, a 
      4-fold improvement over the initial strain achieved by iteratively balancing 
      pathway expression. Including a methylated product in the total, the strain 
      produces 1.3 g/l combined itaconic acids. Further, a regression analysis of the 
      libraries reveals the optimal expression level of CAD as well as pairwise 
      interdependencies between genes that result in increased titer and purity of 
      itaconic acid. This work demonstrates adapting algorithmic design strategies to 
      guide automated yeast strain construction and learn information after each 
      iteration.
CI  - Copyright (c) 2018 International Metabolic Engineering Society. Published by 
      Elsevier Inc. All rights reserved.
FAU - Young, Eric M
AU  - Young EM
AD  - Synthetic Biology Center, Department of Biological Engineering, Massachusetts 
      Institute of Technology, Cambridge, MA 02139, USA; Broad Institute of MIT and 
      Harvard, 415 Main St., Cambridge, MA 02142, USA.
FAU - Zhao, Zheng
AU  - Zhao Z
AD  - DSM Biotechnology Center, PO Box 1, 2600 MA Delft, The Netherlands.
FAU - Gielesen, Bianca E M
AU  - Gielesen BEM
AD  - DSM Biotechnology Center, PO Box 1, 2600 MA Delft, The Netherlands.
FAU - Wu, Liang
AU  - Wu L
AD  - DSM Biotechnology Center, PO Box 1, 2600 MA Delft, The Netherlands.
FAU - Benjamin Gordon, D
AU  - Benjamin Gordon D
AD  - Synthetic Biology Center, Department of Biological Engineering, Massachusetts 
      Institute of Technology, Cambridge, MA 02139, USA; Broad Institute of MIT and 
      Harvard, 415 Main St., Cambridge, MA 02142, USA.
FAU - Roubos, Johannes A
AU  - Roubos JA
AD  - DSM Biotechnology Center, PO Box 1, 2600 MA Delft, The Netherlands.
FAU - Voigt, Christopher A
AU  - Voigt CA
AD  - Synthetic Biology Center, Department of Biological Engineering, Massachusetts 
      Institute of Technology, Cambridge, MA 02139, USA; Broad Institute of MIT and 
      Harvard, 415 Main St., Cambridge, MA 02142, USA. Electronic address: 
      cavoigt@gmail.com.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20180509
PL  - Belgium
TA  - Metab Eng
JT  - Metabolic engineering
JID - 9815657
RN  - 0 (Succinates)
RN  - Q4516562YH (itaconic acid)
SB  - IM
MH  - *Algorithms
MH  - *Gene Library
MH  - Metabolic Engineering/*methods
MH  - *Saccharomyces cerevisiae/genetics/metabolism
MH  - Succinates/*metabolism
OTO - NOTNLM
OT  - Multivariate statistical methods
OT  - Pathway engineering
OT  - Synthetic biology
OT  - Yeast metabolic engineering
EDAT- 2018/05/13 06:00
MHDA- 2019/07/23 06:00
CRDT- 2018/05/13 06:00
PHST- 2018/03/13 00:00 [received]
PHST- 2018/05/04 00:00 [revised]
PHST- 2018/05/04 00:00 [accepted]
PHST- 2018/05/13 06:00 [pubmed]
PHST- 2019/07/23 06:00 [medline]
PHST- 2018/05/13 06:00 [entrez]
AID - S1096-7176(18)30118-6 [pii]
AID - 10.1016/j.ymben.2018.05.002 [doi]
PST - ppublish
SO  - Metab Eng. 2018 Jul;48:33-43. doi: 10.1016/j.ymben.2018.05.002. Epub 2018 May 9.