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Polylactic acid (PLA), a homopolymer of lactic acid (LA), is a bio-derived, biocompatible, and biodegradable polyester. The evolved class II PHA synthase (PhaC1
Ps
6-19
) was commonly utilized in the
de novo
biosynthesis of PLA from biomass. This study tested alternative class I PHA synthase (PhaC
Cs
) from
Chromobacterium
sp. USM2 in engineered
Escherichia coli
for the
de novo
biosynthesis of PLA from glucose. The results indicated that PhaC
Cs
had better performance in PLA production than that of class II synthase PhaC1
Ps
6-19
. In addition, the
sulA
gene was engineered in PLA-producing strains for morphological engineering. The morphologically engineered strains present increased PLA production. This study also tested fused propionyl-CoA transferase and lactate dehydrogenase A (fused Pct
Cp
/LdhA) in engineered
E. coli
and found that fused Pct
Cp
/LdhA did not apparently improve the PLA production. After systematic engineering, the highest PLA production was achieved by
E. coli
MS6 (with
PhaC
Cs
and
sulA
), which could produce up to 955.0 mg/L of PLA in fed-batch fermentation with the cell dry weights of 2.23%, and the average molecular weight of produced PLA could reach 21,000 Da.